SMALL MOLECULES FOR NEURONAL DIFFERENTIATION OF EMBRYONIC STEM CELLS

• US 20110046092A1
• Filed: 12/11/2008
• Published: 02/24/2011
• Est. Priority Date: 12/11/2007
• Status: Active Grant

• Current Assignee: Research & Development Foundation
• Original Assignee: Research & Development Foundation
• Inventors: Preynat-Seauve, Olivier, Suter, David M., Krause, Karl-Heinz
• Granted Patent: US 8,609,413 B2
• Time in Patent Office: Days
• US Class Current: 514/150
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2533/32 Polylysine, polyornithine
• CPC Class Codes: C12N 2533/52 Fibronectin; Laminin
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0697 Artificial constructs associating cells of different lineages, e.g. tissue equivalents blood vessels C12N5/0691

Abstract

A method of preparing neural precursor cells by exposing pluripotent stem cells or neural stem cells to a differentiation agent. The agent is a pyridine analog, which in preferred embodiments is a phenylethynyl-substituted or phenylazo-substituted pyridine. In other embodiments, a method of enhancing neural precursor cell survival is provided in which the survival is enhanced by exposure to the pyridine analog. In further embodiments, a method of preparing neuronal cells is provided in which pluripotent or neural stem cells exposed to the pyridine analog are then incubated without the pyridine analog, resulting in differentiation into neurons, astrocytes and oligodendrocytes. These methods may be used in toxicological screens, e.g., to evaluate the neurotoxicity of a test compound.

Claims

1. A method of preparing one or more neural precursor cells in vitro, comprising exposing substantially or essentially undifferentiated mammalian pluripotent or neural stem cells to an effective amount of a differentiation agent under conditions sufficient to enhance differentiation of the stem cells to neural precursor cells or neurons as compared to differentiation under similar conditions without the differentiation agent, wherein the differentiation agent is a pyridine derivative having the following structure:

2. The method of claim 1, wherein Z₁ and Z₂ are both carbon triply bonded to each other.

3. The method of claim 1, wherein Z₁ and Z₂ are both nitrogen doubly bonded to each other.

4. The method of claim 1, wherein the differentiation agent is:

phenazopyridine;

SIB 1893;

SIB 1757;

2-methyl-6-(phenylethynyl)-pyridine (MPEP);

NSC41777;

6-methyl-3-phenyldiazenylpyridin-2-amine;

2,6-Diamino-3-(4-iodophenylazo)pyridine (U.S. Pat. No. 7,660,000);

phenyldiazenylpyridin-2-amine;

3-(4-chlorophenyl)diazenylpyridine-2,6-diamine;

3-(2-chlorophenyl) diazenylpyridine-2,6-diamine;

3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP);

a physiologically acceptable salt thereof;

or any mixture thereof.

5. The method of claim 1, wherein the stem cells are embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, or embryonic stem cells derived by somatic cell nuclear transfer.

6. The method of claim 1, wherein the stem cells are adult or embryonic neural stem cells.

7. The method of claim 1, wherein the stem cells are mouse, human or primate stem cells.

8. The method of claim 1, wherein neural precursor cells prepared by exposure to the differentiation agent are synchronized.

9. The method of claim 1, wherein undifferentiated cells and non-neural cells are eliminated by exposure to the differentiation agent.

10. The method of claim 5, wherein the stem cells are ES cells.

11. The method of claim 10, wherein the ES cells are obtained from an embryo or a blastocyst.

12. The method of claim 10, wherein the ES cells are obtained from a cell culture comprising undifferentiated ES cells.

13. The method of claim 10, wherein the ES cells are mouse cells.

14. The method of claim 10, wherein the ES cells are human cells.

15. The method of claim 10, wherein the ES cells are monkey cells.

16. The method of claim 10, wherein neural precursor cells prepared by exposure to the differentiation agent are synchronized.

17. The method of claim 10, wherein undifferentiated cells and non-neural cells are eliminated by exposure to the differentiation agent.

18. The method of claim 1, wherein the method further comprises a method of measuring a pharmacological or toxicological property of a test compound comprising contacting said neural precursor cells or neurons with the test compound.

19. The method of claim 18, wherein the neural precursor cells or neurons are comprised in an engineered neural tissue (ENT).

20. The method of claim 18, wherein said property is neurotoxicity.

21. The method of claim 20, wherein cell survival, oxidative stress, or neural function of the neural precursor cells or neurons is measured during or subsequent to said contacting.

22. The method of claim 20, wherein the neural precursor cells or neurons transgenically express a protein marker or tag.

23. The method of claim 22, wherein the protein marker or tag is a luminescent or fluorescent protein.

24. The method of claim 23, wherein the luminescent or fluorescent protein is selected from the group consisting of GFP, eGFP, EBFP, EBFP2, Azurite, mKalama1, ECFP, Cerulean, CyPet, YFP, Citrine, Venus, and YPet.

25. The method of claim 23, wherein the luminescent or fluorescent protein is selected from the group consisting of firefly luciferase (Fluc) and Renilla luciferase (Rluc).

26. The method of claim 23, wherein expression of the luminescent or fluorescent protein is measured optically or via fluorescence activated cell sorting (FACS).

27. The method of claim 18, wherein the method is automated or comprises a high-throughput method.

28. The method of claim 1, wherein the pluripotent or neural stem cells are differentiated into neural precursor cells.

29. The method of claim 1, wherein the pluripotent or neural stem cells are differentiated into neurons.

54. A population of neuronal precursor cells prepare by the method of claim 1.

30. A method of enhancing neural precursor cell survival in vitro, comprising exposing a neural precursor cell to an effective amount of an active agent under conditions sufficient to enhance cell survival of the neural precursor cell as compared to cell survival under similar conditions without the active agent,

wherein the active agent is a pyridine derivative having the following structure:

31-39. -39. (canceled)

40. A method of preparing one or more neuronal cells in vitro, comprising:

a) exposing substantially or essentially undifferentiated mammalian pluripotent or neural stem cells to an effective amount of a differentiation agent under conditions sufficient to enhance differentiation of the stem cells to neural precursor cells as compared to differentiation under similar conditions without the differentiation agent; and

b) incubating the neural precursor cells in the absence of the differentiation agent under conditions sufficient to differentiate the neural precursor cells into neurons, astrocytes or oligodendrocytes;

wherein the differentiation agent is a pyridine derivative having the following structure;

41-53. -53. (canceled)

55. A method of directing the differentiation of pluripotent or neural stem cells to neural cells in vitro, comprising exposing substantially or essentially undifferentiated pluripotent or neural stem cells to an effective amount of a differentiation compound under conditions sufficient to enhance differentiation of the stem cells to neural cells as compared to differentiation under similar conditions without the differentiation compound,

wherein the differentiation compound is a pyridine derivative having the following structure:

56-58. -58. (canceled)

59. A method of treating nervous system damage, comprising administering to a patient in need of such treatment an effective amount of a pyridine derivative having the following structure:

60-66. -66. (canceled)